Description

In eukaryotic replication, DNA polymerase epsilon (Pol ε) is in charge of leading strand synthesis. The catalytic polymerase domain is responsible for correct nucleotide incorporation whereas the exonuclease domain excises misincorporated nucleotides. Importantly, mutations in Pol ε have been implicated in a variety of cancers, including uterine, colorectal, skin, and stomach. These tumor-driving mutants have not yet been well characterized. Here, by using a truncated active form of the enzyme, we first optimize the purification of WT Polε. To improve protein activity and yields, we tested different buffers and chromatography methods such as affinity chromatography using cobalt column and size exclusion chromatography to obtain protein of high purity and activity. We analyzed the effect of various incubation time post induction on the concentration of active enzyme production, as well as the effect of different iron-sulfur cluster concentrations on the activity of the polymerase. This allowed us to identify the most robust strategy for the heterologous expression and purification of active Pol ε. Finally, we investigated the rates of correct incorporation of a single nucleotide in a double-stranded DNA substrate by WT Pol ε by performing burst assay experiments using a rapid chemical quench. We observed an initial burst of product formation associated with single enzyme turnover followed by a slower linear phase corresponding to multiple enzyme turnovers in the steady-state, typical of DNA polymerases. This will allow us to obtain values of Kd of single correct nucleotide incorporation by Pol ε WT exo- as well as single correct and incorrect nucleotide excision by WT exo+ form of the enzyme. Ultimately, this will help us understand the factors that drive genomic instability in human cancer.